Methods of evaluating an immune response to an antigen

ABSTRACT

The present invention incorporates germinal centers (GCs) into three-dimensional (3D) engineered tissue constructs (ETCs). In an embodiment, we have incorporated the GC in the design of an artificial immune system (AIS) to examine immune responses to vaccines and other compounds. Development of an in vitro GC adds functionality to an AIS, in that it enables generation of an in vitro human humoral response by human B lymphocytes that is accurate and reproducible, without using human subjects. The invention also permits evaluation of, for example, vaccines, allergens, and immunogens, and activation of human B cells specific for a given antigen, which can then be used to generate human antibodies. In an embodiment of the present invention the function of the in vitro GC is enhanced by placing FDCs and other immune cells in a 3D ETC; FDCs appear more effective over a longer time (antibody production is sustained for up to about 14 days.

CROSS REFERENCE TO RELATED CASES

This application is a divisional of U.S. application Ser. No.11/642,938, filed Dec. 21, 2006, which issued as U.S. Pat. No. 8,003,387on Aug. 23, 2011 and which claims the benefit of U.S. ProvisionalApplication Ser. No. 60/752,034, filed Dec. 21, 2005, both of which areherein incorporated by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH

This invention was made with government support under contract numberNBCHC060058, awarded by the Defense Advanced Research Projects Agency,issued by the U.S. Army Medical Research Acquisition Activity, andadministered by the U.S. Department of the Interior-National BusinessCenter. The government has certain rights in the invention.

BACKGROUND OF THE INVENTION

We have previously developed functional in vitro germinal centers (GCs)using naïve murine B cells. The model was studied in two dimensions(2-D) in culture plates. In these murine in vitro GCs, immunoglobulin(Ig) class switching, somatic hypermutation, selection of the highaffinity B cells, and affinity maturation were demonstrated. Theseactivities are important to the goal of studying vaccines in vitro. Inthe in vitro GC, follicular dendritic cells (FDCs) serve two mainfunctions: to facilitate T cell-B cell interaction and to potentiate Bcell viability. Both of these functions enable and facilitate activationof specific B cells, antibody production, and differentiation intoplasma cells.

In 1968, Szakal and Hanna (J. Immunol. 101, 949-962; Exp. Mol. Pathol.8, 75-89) and Nossal et al. (J. Exp. Med. 127, 277-290) published thefirst descriptions and electron micrographs of what are now known asfollicular dendritic cells (FDCs). Both groups used ¹²⁵I-labeledantigens and examined autoradiographs of the follicles in rodent spleensor lymph nodes using electron microscopy. Both groups found thatradiolabel persisted on or near the surface of highly convoluted finecell processes of dendritic-type cells with peculiar, irregularlyshaped, euchromatic nuclei. The fine cell processes formed an elaboratemeshwork around passing lymphocytes, allowing extensive cell-cellcontact. Several names have been used for these cells but a nomenclaturecommittee recommended the name “follicular dendritic cell” and theabbreviation “FDC” and these have been generally adopted (Tew et al.(1982) J. Reticuloendothelial Soc. 31, 371-380).

The ability of FDCs to trap and retain antigen-antibody complexes,together with their follicular location, distinguishes them from othercells, including other dendritic cells (DCs). FDCs bearing specificantigens are required for full development of GCs (Kosco et al. (1992)J. Immunol. 148, 2331-2339; Tew et al. (1990) Immunol. Rev. 117,185-211) and are believed to be involved in Ig class switching,production of B memory cells, selection of somatically mutated B cellswith high affinity receptors, affinity maturation, induction ofsecondary antibody responses, and regulation of serum IgG with highaffinity antibodies (Tew et al. (1990) Immunol. Rev. 117, 185-211; Berek& Ziegner (1993) Immunol. Today 14, 400-404; MacLennan & Gray (1986)Immunol. Rev. 91, 61-85; Kraal et al. (1982) Nature 298, 377-379; Liu etal. (1996) Immunity 4, 241-250; Tsiagbe et al. (1992) Immunol. Rev. 126,113-141). Many researchers have worked with FDCs in culture in 2D withthe general idea of mimicking an in vivo GC. An appreciation of theaccessory functions of FDCs and regulation of these functions isimportant to an understanding of fully functional and mature antibodyresponses.

FDC development is B cell-dependent; FDCs are not detectable in, forexample, SCID mice, mice treated with anti-mu (to remove B cells), ormice lacking the mu chain (where B cells do not develop) (MacLennan &Gray (1986) Immunol. Rev. 91, 61-85; Kapasi et al. (1993) J. Immunol.150, 2648-2658). In T cell-deficient mice (e.g., nude mice), FDCs dodevelop, although the development is retarded and the FDCs do not appearto express many FDC markers (Tew et al. (1979) Aust. J. Exp. Biol. Med.Sci. 57, 401-414).

Reconstitution of FDCs in SCID mice occurs best when both B cells and Tcells are adoptively transplanted, suggesting that T cells are alsoinvolved in FDC development (Kapasi et al. (1993) J. Immunol. 150,2648-2658). Disruption of LT/TNF or the cognate receptors disrupts lymphnode organogenesis and interferes with the development of FDC networks(De Togni et al. (1994) Science 264, 703-707; Rennert et al. (1996) J.Exp. Med. 184, 1999-2006; Chaplin & Fu (1998) Curr. Opin. Immunol. 10,289-297; Endres et al. (1999) J. Exp. Med. 189, 159-168; Ansel et al.(2000) Nature 406, 309-314). As summarized by Debard et al. (1999), itis known that a lack of LTα, LTβ, TNFαR1, and LTIβR interferes with thedevelopment of FDC networks (Semin. Immunol. 11, 183-191). B cells arean important source of LTα/β heterotrimers, consistent with dataindicating that FDC development is B cell-dependent (Endres et al.(1999) J. Exp. Med. 189, 159-168; Ansel et al. (2000) Nature 406,309-314; Fu et al. (1998) J. Exp. Med. 187, 1009-1018).

The functional element of a mammalian lymph node is the follicle, whichdevelops a GC when stimulated by an antigen. The GC is an active area ina lymph node, where important interactions occur in the development ofan effective humoral immune response. Upon antigen stimulation,follicles are replicated and an active human lymph node may have dozensof active follicles, with functioning GCs. Interactions between B cells,T cells, and FDCs take place in GCs. Various studies of GCs in vivoindicate that the following events occur there:

-   -   immunoglobulin (Ig) class switching,    -   rapid B cell proliferation (GC dark zone),    -   production of B memory cells,    -   accumulation of select populations of antigen specific T cells        and B cells,    -   hypermutation,    -   selection of somatically mutated B cells with high affinity        receptors,    -   apoptosis of low affinity B cells,    -   affinity maturation,    -   induction of secondary antibody responses, and    -   regulation of serum immunoglobulin G (IgG) with high affinity        antibodies.

Similarly, data from in vitro GC models indicate that FDCs are involvedin:

-   -   stimulating B cell proliferation with mitogens and it can also        be demonstrated with antigen (Ag),    -   promoting production of antibodies including recall antibody        responses,    -   producing chemokines that attract B cells and certain        populations of T cells, and    -   blocking apoptosis of B cells.

While T cells are necessary for B cell responses to T cell-dependentantigens, they are not sufficient for the development of fullyfunctional and mature antibody responses that are required with mostvaccines. FDCs provide important assistance needed for the B cells toachieve their full potential (Tew et al. (2001) Trends Immunol. 22,361-367).

Humoral responses in vaccine assessment can be examined using anartificial immune system (AIS). Accessory functions of folliculardendritic cells and regulation of these functions are important to anunderstanding of fully functional and mature antibody responses.

Important molecules have been characterized by blocking ligands andreceptors on FDCs or B cells. FDCs trap antigen-antibody complexes andprovide intact antigen for interaction with B cell receptors (BCRs) onGC B cells; this antigen-BCR interaction provides a positive signal forB cell activation and differentiation. Engagement of CD21 in the B cellco-receptor complex by complement derived FDC-CD21L delivers animportant co-signal. Coligation of BCR and CD21 facilitates associationof the two receptors and the cytoplasmic tail of CD19 is phosphorylatedby a tyrosine kinase associated with the B cell receptor complex (Carteret al. (1997) J. Immunol. 158, 3062-3069). This co-signal dramaticallyaugments stimulation delivered by engagement of BCR by antigen andblockade of FDC-CD21L reduces the immune responses ˜10- to ˜1.000-fold.

BRIEF SUMMARY OF THE INVENTION

The present invention is directed to an artificial immune system topermit the assessment of allergens, immunogens, immunomodulators,immunotherapies, and potential vaccine agents without administration toanimal subjects, comprising:

-   -   an engineered tissue construct; and    -   at least one three-dimensional artificial germinal center        embedded in or fixed on the engineered tissue construct, said        artificial germinal center comprising:        -   follicular dendritic cells;        -   B cells; and        -   T cells.

The artificial immune system of the present invention can be used inmethods for evaluating the potential reaction of an animal to an agent.Such a method comprises administering an agent to the artificial immunesystem of the present invention and evaluating the B cell and/or T cellresponses to said agent.

The artificial immune system of the present invention can also be usedin methods for producing antibodies specific for an agent. Such a methodcomprises administering an agent to the artificial immune system of thepresent invention and isolating antibodies specific for said agent fromthe artificial immune system. In a similar manner, B cells producingantibodies specific for an agent, or T cells specific for an agent, canalso be isolated from the artificial immune system of the presentinvention. The isolated B cells (which may be monoclonal for the agentin question) can be isolated, cloned and immortalized.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Important receptors and ligands in signaling B cells. The needfor B cell MHC II to present antigen to TCR is well known as is theinvolvement of CD40. Important events include:

-   -   FDCs provide intact antigen to interaction with BCRs and this        antigen-BCR interaction provides a positive signal for B cell        activation and differentiation.    -   FDCs provide a complement derived CD21L for B cell-CD21 and this        interaction with the CD21/CD19/TAPA-1 complex delivers a        positive co-signal for B cell activation and differentiation.    -   FcγRIIB on FDCs bind Ig-Fc in the antigen-antibody complex and        consequently the signal delivered via ITIM in the B cells may be        blocked. (Note that FcγRIIB on the B cell is not engaged). Thus        FDCs minimizes a negative signal to the B cell.    -   FDCs provide IC coated bodies (iccosomes), which B cells find        highly palatable. Iccosomal antigen provides B cells with        antigen to present to T cells.

FIG. 2. FDCs promote the production of NP-specific IgM in cocultureswith naïve B cells. Naïve λ⁺ B cells and FDCs were isolated from naïveC57BL/6 mice and CCG-primed T cells were isolated from C57BL/6 miceimmunized with CGG. ICs were prepared using NP-CGG and anti-CGGhyperimmune mouse serum. ˜1×10⁶ naïve λ⁺ B cells, ˜0.5×10⁶ CGG primed Tcells, ˜0.4×10⁶ FDCs were cocultured in the presence or absence of 100ng NP-CGG in ICs or as free antigen. Culture supernatant fluids werecollected at day 7 and replaced with fresh media. NIP-specific IgMaccumulated in supernatant fluids at day 7 and 14 of cell culture weremeasured using ELISA. All data are representative of three independentexperiments. Panel A shows total NIP-specific IgM and Panel B shows highaffinity NIP-specific IgM antibodies. White columns represent theNIP-specific IgM antibodies generated in the first week and the blackcolumns represent the NIP-specific IgM generated in the second week.Affinity maturation of NIP-specific IgM was estimated by comparing theamount of NIP-specific IgM bound to NIP₁₉-OVA for total and to NIP₅-OVAfor high affinity NIP-specific IgM antibodies. The error bars around themean represent the standard error of the mean for replicate cultures.

FIG. 3. FDCs promote production of NP-specific IgG and affinitymaturation in cocultures with naïve B cells. The same cell cultures usedto study production of NIP-specific IgM in FIG. 1 were used to studytotal and high affinity NIP-specific IgG antibodies. Culture supernatantfluids were collected at day 7 and replaced with fresh media.NIP-specific IgG accumulated in supernatant fluids at day 7 and 14 aftercell culture were measured using ELISA. All data are representative ofthree independent experiments. Panel A shows total NIP-specific IgG andPanel B shows high affinity NIP-specific IgG antibodies. White columnsrepresent the NIP-specific IgG antibodies generated in the first weekand the black columns represent the NIP-specific IgG antibodiesgenerated in the second week. Class switching from IgM to IgG wasestimated by comparing the amount of IgM and IgG generated in the firstvs the second week. Affinity maturation of NIP-specific IgG wasestimated by comparing the amount of NIP-specific IgG bound to NIP₁₉-OVAand NIP₅-OVA. The difference between NIP-specific IgG bound to NIP₁₉-OVAand NIP-specific IgG bound to NIP₅-OVA reflects the affinity maturationof NIP-specific IgG antibodies. The error bars around the mean representthe standard error of the mean for replicate cultures.

FIG. 4. Tissue setting model facts. From Randolph et al. (1998) Science282, 480-3.

FIG. 5. A configuration of the in vitro LTE/GC that incorporates DCs,FDCs, T cells, and B cells on microcarriers.

FIG. 6. Another configuration of the in vitro LTE/GC, which incorporatesDCs, FDCs, T cells, and B cells in an ECM matrix.

FIG. 7. IgG production after 7 days.

FIG. 8. IgG production after 14 days.

FIG. 9. Extensive processes are seen after the FDCs have been oncollagen for about a week.

FIG. 10. To examine somatic hypermutation in the in vitro GCs, we usedPCR to amplify the VH186.2 gene that is used in the mouse to makeanti-NP. The PCR product was cut from an electrophoresis gel, extracted,and cloned; multiple clones were then sequenced. Of 20 readablesequences, 7 had homology to Vh186.2 germline and were designated VH186clones. The sequences have been aligned against the VH186.2germline-encoded gene. Mutations are indicated with the replacednucleotide. Considerable mutation occurred in the variable gene,consistent with somatic hypermutation.

FIG. 11. The number of unique mutations per 10 nucleotide bases plottedagainst base position.

FIG. 12. FDCs after isolation but before positive selection.

FIG. 13. Selected fresh FDCs after isolation but before positiveselection. Note that some FDCs have processes.

FIG. 14. FDCs after positive selection.

FIG. 15. FDCs after positive selection.

FIG. 16. Use of collagen dot pattern to create GC-like zones in vitro.Areas of preferred attachment of the FDC's are spatially limited toprovide borders with “no FDC” zones.

FIG. 17. FDCs were incubated on the Cytodex beads for 24 h and then thelymphocytes were added. 7 d later, IgG production was determined.

FIG. 18. FDCs were incubated on a collagen dot pattern plate for 24 hand then lymphocytes were added. 7 d later, IgG production wasdetermined (this was on a non-tissue culture treated plate).

DESCRIPTION OF THE INVENTION

The present invention is an improvement on previously reported work,incorporating GCs into three-dimensional (3D) engineered tissueconstructs (ETCs). In an embodiment of the present invention, we haveincorporated the GC in the design of an artificial immune system (AIS)to examine immune (especially humoral) responses to vaccines, allergens,immunogens, immunomodulators, immunotherapies, and other agents. In anembodiment of the present invention, development of an in vitro GC addsfunctionality to an AIS, in that it enables generation of an in vitrohuman humoral response by human B lymphocytes that is accurate andreproducible without using human subjects. The invention also permitsevaluation of, for example, vaccines, allergens, immunomodulators,immunogherapies and immunogens, and other agents, and activation ofhuman B cells specific for a given antigen, which can then be used togenerate antibodies. In an embodiment of the present invention thefunction of the in vitro GC is enhanced by placing FDCs and other immunecells in a 3D ETC; FDCs appear more effective over a longer time(antibody production is sustained for up to 14 days).

Embodiments of the present invention comprise placing FDCs in an ETC,such as a collagen cushion, gelatin, hyaluronic acid, small intestinesubmucosa, urinary bladder mucosa, PLGA, hydrogels, plates coated withcollagen, microcarriers, inverted colloid crystal matricies, or othersynthetic or natural extracellular matrix material, where they candevelop in three dimensions. FDCs in the in vivo environment areattached to collagen fibers and do not circulate, as most immune systemcells do. Thus, placing FDCs in, for example, a collagen matrix ought tobe more in vivo-like. In other embodiments, in addition to creating theGC in 3D, a follicle with GC, T cell zones, and B cell zones in thescaffolding provided by the ETC matrix can be developed. Immobile FDCsform a center and the chemokines they secrete may help define the basicfeatures of an active follicle.

Being able to reconstruct follicles where important events forproductive humoral immune responses take place is of importance inassessing vaccines. For example, it is not uncommon to findnon-responders to particular vaccine; such people may be put at riskwhen given a live vaccine. In an embodiment of the present invention,such non-responders can be identified by establishing a model of theirimmune system in vitro and determining their non-responsive or poorlyresponsive state before they were challenged with a live vaccine capableof causing harm. In another embodiment of the present invention,immunomodulators that could convert such poor responders into goodresponders can be identified and formulated for use in vivo. Such anapproach has the potential to reduce vaccine development times and costsand to improve vaccine efficacy and reduce reliance on animal models.

The present invention can also be used for producing antibodies specificfor an agent, B cells that produce antibodies specific for an agent,and/or T cells specific for an agent. In such embodiments, an agent(i.e. a vaccine, an adjuvant, an immunotherapy candidate, a cosmetic, adrug, a biologic, a proinflammatory agent, a chemical compound, anallergen, an immunogen, or an immunomodulator) is administered to theartificial immune system of the present invention. After enough time haspassed for the artificial immune system to produce an immune response tothe agent, antibodies specific for the agent, B cells that produceantibody specific for the agent, and/or T cells specific for the agentare isolated from the artificial immune system. The isolated B cellsthat produce antibodies (optionally monoclonal antibodies) specific forthe agent and the isolated T cells specific for the antigen can becloned and immortalized. Methods for immortalizing B cells and T cellsare well known to one of skill in the art. See, for example, Aguirre etal., (2000) J. Virol. 74(2):735-743; and Robek et al. (1999) J. Viol.73(6):4856-4865.

In addition, some therapeutic agents and industrial chemicals are toxicto the immune system and in other embodiments an in vitro immune systemcomprising in vitro germinal centers could be used to assessimmunotoxicity and the effects of allergens in the context of a modelhuman immune system. The present invention can also be used to assesstherapeutic agents that could convert immune responders tonon-responders, which would be invaluable for the treatment ofantibody-mediated autoimmune disorders.

It has been observed that treatment of animals with LTβR-Ig disruptsFDCs' ability to retain immune complexes (ICs), which has beenattributed to competition for B cell-derived LTα/β heterotrimers, thusreducing the ability to stimulate FDCs (Mackay & Browning (1998) Nature395, 26-27). Under these conditions, FDCs appear to lose their activatedphenotype and ICs tend to disappear. We have obtained similar results invitro in and have shown that FDC function in promoting antibodyproduction and blocking B cell apoptosis is adversely affected when thecells are incubated with LTβR-Ig.

Our data indicate that FDCs do not die as a consequence of lack ofstimulation by LT or TNF and that they can exist for long periods in aresting state. Indeed, it has been reported that human FDCs survive inthe absence of B cells for months in cell culture without proliferationalthough the antigenic phenotype (positive for DRC-1, CD21, CD23, CD35)disappears after only a few days (Tsunoda et al. (1990) Virchows. Arch.B Cell. Pathol. Incl. Mol. Pathol. 59, 95-105). Murine FDCs can alsosurvive for months in culture and when present in an in vitro GC theymaintain T cells, B cells, and a functioning immune system (Qin et al.(1999) J. Immunol. Methods 226, 19-27). We found that purified murineFDCs can survive for long periods (at least 6 weeks) in the absence ofother cells. However, it appears that these resting FDCs can beactivated by stimulation of the FDC with T cells and B cells, togetherwith ICs and complement.

We have found that FDCs express CD40 and the level on freshly isolatedFDCs appears to be higher than the level on B cells, suggesting that GCT cells may have an attractive receptor for their CD40L. Engagement ofCD40 is known to activate B cells, dendritic cells, and macrophages.Given the importance of CD40 in activation of these immunologicallyrelevant cells, FDC-CD40 may also be involved in FDC activation (Caux etal. (1994) J. Exp. Med. 180, 1263-1272). Some FDC markers (e.g., CD23)appear to be T cell-dependent and engagement of FDC-CD40 by CD40L on Tcells in active GCs is likely important to full expression of the activeFDC phenotype. Expression of the FDC-M2 antigen and CD21L arecomplement-dependent. The FDC-M2 antigen is now known to be a fragmentof C4, which binds covalently to ICs on FDCs (Marie Kosco-Vilbois,personal communication). Similarly, a fragment of C3 binds covalently toICs and forms the CD21L (Qin et al. (1998) J. Immunol. 161, 4549-4554).Thus, it appears that development, maturation, and full activation ofFDCs requires B cells, T cells, and complement.

We have also examined FDC accessory molecules and accessory functions.FDCs, B cells, and T cells are clustered together in GCs and cell-cellcontact appears to be important because we have yet to find an FDCaccessory activity that will work well across a semi-permeable membrane(Wu et al. (1996) J. Immunol. 157, 3404-3411; Tew et al. (1997) Immunol.Rev. 156, 39-52). FDCs may produce important cytokines but clearly cellsurface molecules are important in these cell-cell interactions. Areview of some of our data was previously published (Tew et al. (2001)Trends Immunol. 22, 361-367).

Immunogens are quickly converted into immune complexes (ICs) byantibodies persisting in immune animals from prior immunization(s) andICs form in primary responses as soon as the first antibody is produced.These ICs are trapped by FDCs and this leads to GC formation. Immunecomplexes are poorly immunogenic in vitro, yet minimal amounts ofantigen (converted into ICs in vivo) provoke potent recall responses.

Our results indicate that FDCs render ICs highly immunogenic. In fact,in the presence of FDCs, ICs are more immunogenic than free antigen (Tewet al. (2001) Trends Immunol. 22, 361-367). A high density of FcγRIIB onFDCs bind Ig-Fc in the IC and consequently the ITIM (immunoreceptortyrosine-based inhibitory motif) signal delivered via B cell-FcγRIIB maybe blocked. Antigen-antibody complexes cross-linking BCRs initiate thisinhibitory signal and FcγRIIB on B cells. BCR is not cross-linked with Bcell FcγRIIB in the model and thus a high concentration of FcγRIIB onFDCs minimizes the negative signal to the B cell. In addition, FDCsprovide IC-coated bodies (iccosomes), which B cells find highlypalatable. The iccosome membrane is derived from FDC membranes that haveantigen, CD21L, and Ig-Fc attached. Iccosomes bind tightly to B cellsand are rapidly endocytosed (Szakal et al. (1988) J. Immunol. 140,341-353). Binding of BCR and CD21 of the B cell to the iccosomalantigen-CD21L-Ig-Fc complex is likely important in the endocytosisprocess. The B cells process this FDC-derived antigen, present it, andthus obtain T cell help (Kosco et al. (1988) J. Immunol. 140, 354-360).Thus, these ligand-receptor interactions help stimulate B cells andprovide assistance beyond that provided by T cells.

Another important molecule associated with FDC function is CD23. Wefound that serum IgE is suppressed in CD23 transgenic mice where highlevels of CD23 are expressed on FDCs and B cells and some T cells(Payet-Jamroz et al. (2001) J. Immunol. 166, 4863-4869). When purifiedtransgenic B lymphocytes were compared with controls in B cellproliferation and IgE synthesis assays in vitro, the two wereindistinguishable. Similarly, studies of lymphokine production suggestedthat T cell function in the transgenic animals was normal. However,adoptive transfer studies indicated that IgE production was dramaticallysuppressed when normal lymphocytes were used to reconstitute transgenicmice, which would have high levels of CD23 on the radioresistanttransgenic FDCs. Furthermore, when FDCs were isolated from thetransgenic mice, FDC-dependent IgG production in cell culture was nearnormal but IgE production was dramatically reduced, suggesting that highlevels of CD23 on FDCs can selectively suppress IgE responses(Payet-Jamroz et al. (2001) J. Immunol. 166, 4863-4869). Interestingly,IL-4 induces CD23 on B cells but does not appear to induce CD23 on FDCs.However, in mice immunized using complete Freund's adjuvant (CFA), thelevel of CD23 on the FDCs is dramatically increased (Maeda et al. (1991)In “Dendritic Cells in Lymphoid Tissues.” Y. Imai, J. G. Tew & E. C. M.Hoefsmit, eds. Elsevier Science, Amsterdam, pp. 261-269). If CD23 iselevated, then unoccupied CD23 on FDCs may bind B cell surface-IgE andthis could result in an inhibition of IgE production. Thus, FDCs bearinghigh levels of CD23 may selectively down regulate specific IgE responsesand this may explain why IgE responses in CFA-immunized animals arerelatively low. Furthermore, the association with CFA suggests CD23 onFDCs may be regulated by Th-1 lymphokines.

ICs trapped by FDCs lead to GC formation. GC formation is involved inthe production of memory B cells, somatic hypermutation, selection ofsomatically mutated B cells with high affinity receptors, affinitymaturation, and regulation of serum IgG with high affinity antibodies(Tew et al. (1990) Immunol. Rev. 117, 185-211; Berek & Ziegner (1993)Immunol. Today 14, 400-404; MacLennan & Gray (1986) Immunol. Rev. 91,61-85; Kraal et al. (1982) Nature 298, 377-379; Liu et al. (1996)Immunity 4, 241-250; Tsiagbe et al. (1992) Immunol. Rev. 126, 113-141).

The GC is generally recognized as a center for production of memory Bcells; we have found that cells of the plasmacytic series are alsoproduced (Kosco et al. (1989) Immunol. 68, 312-318; DiLosa et al. (1991)J. Immunol. 1460, 4071-4077; Tew et al. (1992) Immunol. Rev. 126, 1-14).The number of antibody-forming cells (AFCs) in GCs peaks during an earlyphase (about 3 to about 5 days after secondary antigen challenge) andthen declines. By about day 10 when GCs reach maximal size, there arevery few AFCs present (Kosco et al. (1989) Immunol. 68, 312-318). Duringthe early phase, GC B cells receive signals needed to become AFCs. TheGC becomes edematous and the AFCs leave and we find them in the thoracicduct lymph and in the blood. These GC AFCs home to bone marrow wherethey mature and produce the vast majority of serum antibody (DiLosa etal. (1991) J. Immunol. 1460, 4071-4077; Tew et al. (1992) Immunol. Rev.126, 1-14; Benner et al. (1981) Clin. Exp. Immunol. 46, 1-8). In thesecond phase, which peaks about 10-14 days after challenge, GCs enlarge,and the memory B cell pool is restored and expanded. Thus, production ofB memory and fully functional and mature antibody responses appears torequire GCs and FDCs.

Potentiating B cell viability can be done with or without FDCs presentto enhance in vitro GC efficacy. A method is to add fibroblasts or otherstromal cells, such as synovial tissue-derived stromal cell lines, theeffects of which are to prolong B cell viability in vitro throughcell-cell co-stimulation (e.g., Hayashida et al. (2000) J. Immunol, 164,1110-1116). Another soluble agent that has been shown to increase naïveand memory B cell viability is reduced glutathione (GSH), perhapsthrough anti-oxidant activity (see Jeong et al. (2004) Mol. Cells 17,430-437). Although Jeong et al. did not see enhanced viability of GC Bcells, they did significantly enhance naïve and memory B cells withfibroblasts and GSH, suggesting that peripheral B lymphocytes can beused to populate the in vitro GC. Other soluble factors, such as IL-4,CD40L and anti-CD40 have been shown to potentiate B cell viability (L.Mosquera's work and M. Grdisa (2003) Leuk. Res. 27, 951-956). Ancillaryfactors and cells that increase B cell viability with or without FDCswill enhance in vitro GC performance.

Compared with other leukocytes, FDCs have received little attention. Anunderstanding of FDCs is important to an understanding of B lymphocytematuration and antibody production. This lack of information on FDCs islikely because these cells are rare and fragile. Knowledge of moretypical leukocytes has been derived largely from in vitro studies ofisolated populations.

We have developed techniques to isolate and work with FDCs andFDC-lymphocyte interactions can now be studied in vitro along withantigen, antigen-antibody complexes, and polyclonal B cell activators.FDCs with appropriate ICs have remarkable accessory activity wheninteracting with B cells and can:

-   -   block apoptosis in B cells (Schwarz et al. (1999) J. Immunol.        163, 6442-6447; Qin et al. (1999) J. Immunol. Methods 226,        19-27),    -   block ITIM (immunoreceptor tyrosine-based inhibitory motif)        signaling in B cells stimulated by ICs (Aydar et al. (2004)        Eur. J. Immunol. 34, 98-107),    -   promote B cell proliferation stimulated by antigen or mitogen        (Burton et al. (1993) J. Immunol. 150, 31-38).    -   promote recall responses (Tew et al. (2001) Trends Immunol. 22,        361-367),    -   induce virgin B cells to produce IgM and promote class switching        to IgG (Kraal et al. (1982) Nature 298, 377-379; Liu et        al. (1996) Immunity 4, 241-250; Aydar et al. (2005) J. Immunol.        174, 5358-5366), and    -   promote somatic hypermutation and the development of high        affinity antibodies (Aydar et al. (2005) J. Immunol. 174,        5358-5366).

These are important features of the humoral immune response.

In vivo FDCs exist in networks linked to collagen and collagenassociated molecules. This linkage allows networks of FDCs to remainstationary while B cells and T cells move in and out of contact with theFDCs and associated antigen. This arrangement has been reconstructed inthe in vitro GCs of the present invention.

We have established that FDCs have an ability to attach to collagen type1, collagen type IV, laminin, biglycan, fibronectin, and hyaluronicacid. Furthermore, we have established that FDCs attached to collagenreestablish a reticulum with interconnecting processes. This ability toattach to collagen and collagen associated molecules contrasts withtheir lack of ability to attach directly to plastic or glass. Our dataindicate that antibody responses are improved when the FDCs are adheredto collagen and collagen-associated molecules.

Vaccination Site Model. Dendritic cells (DCs) are among the most potentantigen-presenting cells (APCs) and are the only known cell type withthe capacity to stimulate naïve T cells in a primary immune response.Peripheral blood monocytes are widely accepted as a reliable source ofprecursor cells for DC generation in vitro. Such monocyte-derived DCs(mo-DCs) posses the overall phenotype and antigen-presenting abilitiesfound in DCs in vivo.

A common generation technique for mo-DCs is based on using the cytokinesGM-CSF and IL-4 for 5 days, leading to cells with an immature phenotype.After antigen priming for a subsequent 2 days, mo-DCs increase theirco-stimulatory and antigen-presenting capabilities to a state calledmaturation.

Interestingly, Randolph et al. found that the likely naturally occurringprocess of monocyte transendothelial migration induces a process ofdifferentiation into DCs in just 2 days, without addition of exogenouscytokines. This process starts with monocytes traversing a monolayer ofendothelial cells in the luminal to abluminal direction, followed by areverse transmigration to the luminal surface after a period of 48 hr ofresting (interaction) within the extracellular matrix (susceptible ofcontaining specific antigens).

In an embodiment of the present invention, the vaccination site modelcomprises a monolayer of endothelial cells (human umbilical veinendothelial cells, HUVECs) grown to confluency over a bovine type Icollagen matrix (cushion). Other vaccination site models can also beemployed, using various ECM materials instead of collagen. Inembodiments of the present invention, the ECM can be in a cushion or amembrane configuration or an endothelium grown over a polycarbonate orother membrane (e.g., a Transwell). The whole monocyte differentiationprocess resembles what is believed to occur in vivo where naturallyoccurring diapedesis of monocytes into the tissues ends up with thedevelopment of tissue-resident macrophages and migratory dendritic cellsescaping to the lumen of the lymphatics by traversing endothelial cellsin the abluminal to luminal direction. In other embodiments, DCmaturation can be achieved based on the presence of stimuli embedded inthe matrix.

We have developed an in vitro system for the generation of immature DCsfrom migratory peripheral blood monocytes. In an embodiment of theinvention, the system comprises a collagen membrane sealed on each sideby a confluent monolayer of endothelial cells. The assembly of this invitro vaccination site (VS) in an integrated bioreactor allows thegeneration of a bicameral device, with independent liquid flow. Theupper chamber contains continuously circulating monocytes and the lowerchamber receives the migratory immature DCs ready to be antigenicallyprimed in situ. After a defined period, antigenically activated mo-DCscan be relocalized (e.g., by means of slow flow or chemokine attraction)to reside in a pre-established lymphoid tissue equivalent (LTE) forinduction of specific immune responses. These mo-DCs will induce animmune response in the LTE that also contains the GC.

EXAMPLES

Unless otherwise indicated, all culture conditions were replicated (3-6replicates depending on power calculations) and blocking antibodies wereused over a range of concentrations (typically, ˜1, ˜10, ˜100 μg/mL) andexperiments were repeated to establish reproducibility. Typically, thedose of antigen in the antigen-antibody complexes is ˜10 to ˜50 ng/mLand these preformed antigen-antibody complexes are at slight antigenexcess, where the stimulatory activity is optimal. The levels ofantibody (anti-NIP, anti-TT, or total IgG) are measured using an ELISA(expressed as ng antibody/mL).

The frequency of B cells with a given antigen specificity can bedetermined with a modified ELISPOT assay, as described by Crotty et al.(2004) J. Immunol. Meths. 286, 111-122. Briefly, B cells isolated fromthe AIS will be stimulated for approximately 5 h with plate-boundantigen in an ELISPOT plate. Activated B cells that are specific for theparticular antigen will secrete antibody, which will be captured on theplate-bound protein. Captured antibody can be detected in a colorimetricassay, and the number of spots provides a sensitive determination of thefrequency of responding cells. A similar ELISPOT-based approach forsecreted cytokines can also be used the estimate the number ofantigen-specific T cells generated within an AIS. In another embodiment,intracellular labeling for cytokines produced following antigen-specificstimulation can provide a similar readout. For well-defined antigens,such as tetanus toxoid, the use of tetrameric complexes of MHC moleculeswith specific peptide can be used to determine the frequency ofantigen-specific T cells by direct detection of the T cell receptoritself.

Direct analysis of activated B and T cells can also be performed byisolating the lymphocytes from the engineered tissue construct (ETC)matrix at different times following antigen encounter. Different ETCmaterials will require differing approaches to dissociate cells from amatrix. For example, collagenase can be used to disrupt a collagenscaffold.

A feature of B and T cell activation is that the cells rapidlyproliferate following antigen encounter. To examine the strength of thelymphocyte response, B and T cell proliferation can be tracked bypre-labeling the cells with the fluorescent dye CFSE prior to theirintroduction into the lymphoid tissue equivalent (LTE), which can bethought of as an in vitro lymph node. CFSE is a stable, long-livedmolecule that binds cytoplasmic proteins via an enzymatic reaction. Thisdivision-sensitive dye is equally distributed amongst daughter cellsfollowing cell division; thus, each divided cells will have half theCFSE fluorescence intensity of the parent cell. By flow cytometricanalysis, up to about 8 to about 10 cell divisions can be detectedwithin a population of proliferating cells.

Lymphocyte activation is also associated with changes in the expressionof membrane proteins that regulate B and T cell function. Acharacteristic of naïve B cell activation is the switch in expression ofsurface IgM to other antibody classes (especially IgG). Additionally,upregulated expression of surface MHC and accessory molecules, such asCD54, CD58, CD80, and CD86, are suggestive of B cell activation, andincreased expression of surface CD27 marks the acquisition of a memoryphenotype in B cells. T cell activation is associated with alteredexpression of molecules that regulate their migration (CD 11a, CD62L)and activation (CD28, CD25). Changes in the expression pattern of eachof these surface molecules can be monitored using standard flowcytometry techniques and commercially available antibodies (e.g., thosefrom BD Pharmingen, Calif.).

Production of soluble growth factors can be used to gauge the inductionof antigen-specific lymphocyte responses. Secreted cytokines, including,but not limited to, IL-2, IFN-γ, TNF-α, IL-4, IL-6, and IL-10, can bedetected following antigen encounter. Expression of certain cytokineprofiles, such as IL-4 and IL-10 that are expressed by only particular Tcell subpopulations, can provide clues to the quality of the adaptiveresponse being generated. Current, commercially available reagents allowfor the detection of soluble cytokines at concentrations in the pg/mLrange.

Generation of adaptive immune responses within the lymphoid tissueequivalent (LTE) of an AIS can be examined at about 7 to about 14 dfollowing antigen administration, the time typically required forinduction of measurable protective immunity during in vivo and in vitroresponses. Changes in the expression pattern of soluble proteins thatare indicative of B and T cell activation/differentiation can beexamined in supernatants harvested from the LTE. Specifically, B cellactivation triggers production of secreted antibody molecules that canbe quantitated by ELISA using commercially available reagents (e.g.,those from Bethyl Laboratories, Tex.). This sensitive technique can beused to detect class switching, an important trait of B cellmaturation/differentiation, by examining the expression of different Igclasses (IgM, IgG, etc.). To determine antigen-specific antibodyproduction, whole protein can be used to capture specific antibody in anELISA. For example, in the well-established NP experimental model, NIP-5and NIP-19 can be used to specifically detect the production ofantibodies against NP with high and high/low affinities, respectively.

Example 1

Animals and Immunization. Normal 8 to 12 wk old C57BL/6 mice can bepurchased from the National Cancer Institute (Frederick, Md.) or TheJackson Laboratory (Bar Harbor, Me.). The mice can be housed in standardplastic cages with filter tops and maintained under specificpathogen-free conditions. Food and water can be supplied ad libitum. CGG(chicken gamma globulin)-primed T cells were obtained after immunizationwith 20 μg CGG (Pel-Freez Biologicals, Rogers, Ark.) and ˜5×10⁸heat-killed Bordetella pertussis precipitated in aluminum potassiumsulfate (A7167, Sigma), as described previously (5,28). The mice weregiven a booster immunization 2 weeks later with ˜50 μg CGG i.p. and by˜5 μg CGG s.c. injection into the front legs and hind footpads.

Example 2

Antibodies and Reagents. Mouse CD45R (B220) MicroBeads, mouse CD90(Thy1.2) MicroBeads, anti-biotin MicroBeads, and MACS LS columns can bepurchased from Miltenyi Biotec GmbH (Auburn, Calif.). Biotin-labeled ratanti-mouse κ can be purchased from Zymed (San Francisco, Calif.).Alkaline phosphatase-labeled goat anti-mouse IgG (H+L), and alkalinephosphatase-labeled goat anti-mouse IgM can be obtained from, e.g.,Kirkegaard & Perry Laboratories (Gaithersburg, Md.). Anti-mouse FDC(FDC-M1) and anti-mouse CD21/CD35 can be purchased from, e.g.,Pharmingen (San Diego, Calif.). NIP₁₉-OVA(4-hydroxy-3-ioda-5-nitrophenylacetyl ovalbumin with 19 NIP groups/OVA),NIP₅-OVA (with 5 NIP groups/OVA), and NP₃₀-CGG can be obtained from,e.g., Biosearch Technologies (Novata, Calif.). Rat anti-mouse CD40 canbe obtained from, e.g., Southern Biotechnology Associates, Inc.Low-tox-m rabbit complement can be purchased from, e.g., CedarlaneLaboratories Limited (Westbury, N.Y.); heat inactivation wasaccomplished by incubating the complement in a water bath at 56° C. for˜30 min. NP-CGG-anti-CGG ICs were prepared by incubating the antigen andantibody for 2 h at 37° C. at final ratio of 1 ng/ml NP-CGG to 6 ng/mLof mouse anti-CGG. The anti-CGG was obtained from hyperimmunized micewith anti-CGG IgG levels in excess of 1 mg/ml. In certain experimentscomplement-bearing ICs were made using low-tox-m rabbit complement at1:12 dilution during the 2 h incubation. Anti-CD21/35 was converted intoF(ab′)₂ fragments using the Pierce (Rockford, Ill.) ImmunoPure F(ab′)₂preparation kit (Cat. #44888). Anti-CD23 (clone B3B4) was provided byDr. Daniel Conrad.

Example 3

FDC Isolation. FDCs were isolated from lymph nodes (axillary, lateralaxillary, inguinal, popliteal, mesenteric, and paraaortic) of normal,young adult mice as described previously (5,28). Briefly, one day beforeFDC isolation the mice were exposed to whole body irradiation toeliminate most T and B cells (1000 rads, using a ¹³⁷Cs source) (Kosco etal. (1992) J. Immunol. 148, 2331-2339). Lymph nodes were collected andeach lymph node capsule was opened using two 26-gauge needles. The lymphnodes were then placed in an enzyme cocktail consisting of 1 mlcollagenase D (16 mg/mL, C-1088882, Roche), 0.5 mL DNaseI (5000units/mL, D-4527, Sigma), and 0.5 mL DMEM, supplemented with 20 mMHEPES, 2 mM glutamine, 50 μg/mL gentamicin, and MEM non-essential aminoacids (GIBCO). After 30 min at 37° C. in a CO₂ incubator, the medium andreleased cells were removed and transferred to a 15 mL conicalcentrifuge tube containing 5 mL DMEM with 20% FCS and placed on ice. Theremaining tissue was subjected to a second 30 min. digestion in a freshaliquot of enzyme mixture and the cells were collected as before.Isolated cells were washed and then incubated with a rat anti-mouse FDCspecific antibody (FDC-M1) for 45 min on ice. The cells were washed andincubated with 1 μg biotinylated anti-rat Ig specific for κ light chainfor 45 min on ice. The cells were then incubated with 40 μL anti-biotinMicroBeads (Miltenyi Biotec) added to 360 μL MACS buffer for 15-20 minon ice. The cells were layered on a MACS LS column pre-wetted with 1 mlMACS buffer and washed with 10 ml of ice-cold MACS buffer. The LS columnwas removed from the VarioMACS and the bound cells were released with 10mL MACS buffer. Approximately 85 to 95% of these cells express the FDCphenotype, FDC-M1⁺, CD40⁺, CR1&2⁺, and FcγRII⁺ (Sukumar et al.,unpublished). Human FDCs can be isolated using positive selection withthe FDC specific mAb HJ2, as previously described (Fakher et al. (2001)Eur. J. Immunol. 31, 176-185).

Example 4

Cell Cultures for Analysis of AID (activation-induced cytidinedeaminase). Lymphocytes (˜4×10⁶) were co-cultured with ˜1.6×10⁶ FDCs in48-well culture plates (CoStar; Cambridge, Mass.) for about ˜2 d at 37°C. in a 5% CO₂ atmosphere. The wells contained ˜1 mL/well of completemedium (DMEM, supplemented with 10% FCS, 20 mM Hepes, 2 mM glutamine, 50μg/mL gentamicin, and MEM-nonessential amino acids). LPS at 10 ng/mL(L-2387, Sigma) or 100 ng/mL anti-CD40+10 ng/mL IL-4 (R&D Systems,Minneapolis, Minn.) were used to stimulate the lymphocytes. Sub-optimallevels of LPS, anti-CD40+IL-4 were used because FDC co-stimulatoryactivity was most apparent at sub-optimal concentrations of the primarysignal. The influence of FDCs was still apparent at higherconcentrations of the primary signal but the differences were smallerand more difficult to study. After 48 h, cells were harvested and lysedusing TRIzol (Invitrogen) and total RNA was extracted, following themanufacturer's protocol. In some experiments, λ⁺ B cells and CGG-primedT cells and NP-CGG+anti-CGG immune complexes were cultured in thepresence or absence of FDCs for 72 h. At the end of 72 h, B cells wereisolated using anti-B220 MicroBeads and the MACS system. Total RNA from˜2×10⁶ B cells was extracted using Trizol.

Example 5

Quantitative Reverse Transcriptase PCR analysis. The mRNA levels for AID(activation-induced cytidine deaminase) were measured using quantitativereverse transcriptase PCR (qRT PCR). The 18s rRNA level was used as aninternal control to normalize the expression levels of AID. PCRreactions were performed in 96-well thin-wall PCR plates covered withtransparent, optical-quality sealing tape (Bio-Rad). Amplifications wereperformed using the One Step RT-PCR kit (Applied Biosystems) under thefollowing conditions: 48° C. for 30 min (cDNA synthesis), initialdenaturation at 95° C. for 10 min, followed by 40 cycles of denaturationat 95° C. for 15 s and a combined annealing/extension step at 60° C. for1 min. Data analysis was performed using the iCycler iQ software(BioRad). Finally, differences in mRNA expression levels were calculatedusing the ΔΔC_(T) method (Livak & Schmittgen (2001) Methods 25,402-408). PCR efficiency was determined to be close to 100% byperforming multiple standard curves using serial mRNA dilutions. Anamplification cycle threshold value (C_(T) value), defined as the PCRcycle number at which the fluorescence signal crosses an arbitrarythreshold, was calculated for each reaction. The fold change betweenmRNA expression levels was determined as follows: Fold change=2^(ΔΔCT),where ΔΔC_(T)=(C_(T GoI)−C_(T Hk)) Sample−(C_(T GoI)−C_(T Hk)) Control(C_(T)=cycle threshold, GoI=gene of interest, and Hk=house keepinggene).

Example 6

Purification of Naïve B Cells. Single cell suspensions were prepared bygrinding lymph nodes from naïve mice between the frosted ends of twosterile slides in complete medium (DMEM supplemented with 10% FCS, 20 mMHepes, 2 mM glutamine, 50 μg/mL gentamicin, and MEM-nonessential aminoacids). The suspended cells were centrifuged (5 min., 1000 rpm, 4° C.)and resuspended in complete medium. The κ+λ-positive B cells (total Bcells) were positively selected using anti-B220-bearing MicroBeads.Briefly, the lymphocytes were incubated with 40 μL anti-B220 MicroBeads(diluted 1:10 in MACS buffer) for 15-20 min on ice. The cells werelayered on a MACS LS column pre-wetted with 1 ml MACS buffer and washedwith 10 mL ice-cold MACS buffer. The LS column was removed from theVarioMACS and the bound cells were released with 10 mL MACS buffer,washed, and used as κ+λ-positive B cells. Anti-NP antibodies in C57BL/6mice predominantly have λ light chains (Jack et al. (1977) Eur. J.Immunol. 7, 559-565; Reth et al. (1978) Eur. J. Immunol. 8, 393-400) andwe reasoned that the NP response would be enhanced if λ-positive naïve Bcells were enriched in culture. To obtain the λ-positive naïve B cells,we removed κ-positive B cells using 10 μg κ light chain-specificbiotinylated rat-anti-mouse mAb for 45 min on ice and trapped theκ-positive B cells on a MACS column with anti-biotin MicroBeads(Miltenyi Biotec). We reasoned that B220-positive cells in the flowthrough would express the λ chain and they were isolated usinganti-B220, as described above. Naïve B cells express membrane IgM andthe presence of IgM on our naïve B cell population was confirmed by flowcytometry. Single-cell suspensions of lymph node cells from normal micewere triple-labeled with FITC B220, PE-conjugated anti-mouse IgM, andbiotin-labeled rat anti-mouse λ. The results indicated that about 95% ofour B cells expressed κ rather than λ light chain. However, nearly 98%of the cells that expressed λ light chains were IgM-positive, which isexpected of B cells in the naïve state. Serum anti-NIP levels in thesedonor mice were too low to measure (<1 ng/mL), again supporting thenaïve nature of the NIP-specific B cells. The same approach can be usedto obtain naïve human B cells from PBL; the markers will be IgM-positiveand CD19-positive.

Example 7

Isolation of CGG-Primed T Cells. CGG-primed lymphocytes were obtainedfrom draining lymph nodes of CGG-immunized mice a week or more after theCGG booster. Lymph nodes were surgically removed and ground between thefrosted ends of two sterile slides. The cells were washed and incubatedwith 40 μL mouse anti-CD90 (Thy1.2) MicroBeads (diluted 1:10 in MACSbuffer) for ˜45 min on ice then layered on a MACS LS column pre-wet with1 ml MACS buffer and washed with ˜10 mL ice-cold MACS buffer. The LScolumn was removed from the VarioMACS and the bound cells were collectedas above. TT (tetanus toxoid)-primed T cells from seropositive humanscan be obtained with anti-CD2.

Example 8

In vitro GC reactions and the Anti-NIP antibody Response. In vitro GCreactions were set up by co-culturing naïve λ positive B cells (˜10×10⁵cells/mL), FDCs (˜4×10⁵ cells/mL), and CGG-primed T cells (˜5×10⁵cells/mL), with NP-CGG+anti-CGG ICs (100 ng NP-CGG/well) in 48-wellculture plates (CoStar; Cambridge, Mass.). The wells contained 1 mL/wellcomplete medium (DMEM, supplemented with 10% FCS, 20 mM Hepes, 2 mMglutamine, 50 μg/mL gentamicin, and MEM-nonessential amino acids). ICswere prepared using NP-CGG and anti-CGG serum, and were used tostimulate the lymphocytes. The cultures were incubated at 37° C. in a 5%CO₂ atmosphere. Supernatant fluids were harvested on days 7 and 14 andwere assayed for NIP-specific low and high affinity IgM and IgGantibodies, using a solid phase ELISA. Each experimental group was setup in triplicate.

Example 9

ELISA for Anti-NIP and Affinity. The relative affinities of anti-NIPantibodies were determined using an ELISA with OVA coupled to NIP atdifferent ratios, respectively, NIP₁₉-OVA and NIP₅-OVA. NIP has higheraffinity for anti-NP antibodies than NP and NIP was used for this reason(44,45). Briefly, flat-bottom 96-well ELISA plates (Falcon; BectonDickinson, Calif.) were coated with 100 μg/mL NIP₅-OVA or NIP₁₉-OVA inPBS at 4° C. overnight. After washing the plates three times with 1×PBScontaining 0.1% Tween 20, the plates were blocked with BSA (5%, 2 h,room temperature). Supernatant fluids from the cultures were then addedto the plates at a starting dilution of 1:2 for wells with low responsesand incubated at 4° C. overnight. Alkaline phosphatase-conjugated goatantibody specific for mouse IgM or IgG was added and incubatedovernight. Alkaline phosphatase activity was visualized using a pNPPphosphatase substrate kit (Kirkegaard & Perry Laboratories, Md.) andoptical densities were determined at 450 nm. Standard curves for IgM orIgG were established by incubating the plates with 100 μg/mLaffinity-purified goat anti-mouse IgM or IgG (Sigma, Saint Louis, Mo.).The plates were then washed and incubated with two-fold dilutions ofmouse IgM or IgG (Sigma) starting at 100 ng/mL and the plates wereincubated at 4° C. overnight. A standard curve was run on each plate andconcentrations of anti-NIP IgM or IgG antibodies were calculated bycomparison to standard curves in the linear dose range. The relativeaffinity of the antibodies was indicated by the level of antibody usingNIP₁₉-OVA, which measures both high and low affinity anti-NIP versusNIP₅-OVA, which indicates only high affinity anti-NIP.

Example 10

Statistical Analysis. For analysis of ELISA readings, at test(two-tailed distribution) was used. In some experiments, up to 5different comparisons were made and a p value of less than 0.01 wasrequired to account for multiple comparisons. The 2^(−ΔΔC) _(T) methoddescribed in Livak & Schmittgen (2001) (Methods 25, 402-408) was used toanalyze real-time quantitative PCR results.

Example 11

Histochemical procedures. A chapter, entitled “Use of monoclonalantibodies in immunocytochemistry at the light and electron microscopiclevels” (Szakal et al. (1986). In Monoclonal Antibodies: HybridomaTechniques. L. B. Schook, ed. Marcel Dekker, Inc, New York, pp. 229-263)describes these in detail. Biotinylated probes allow the use of HRPavidin and allow both light- and electron microcopic-level studies.

Example 12

Studies on in vitro GCs. Promotion of NIP-specific IgM responses in invitro GCs. Stimulation of antigen-specific B cells and class switchingtakes place in GCs; FDCs may enhance IgM responses and Ig classswitching. To assess this, we isolated naïve, IgM-expressing B cellswhere a switch from producing IgM to IgG could be easily monitored.NIP-specific antibody responses were initiated in the in vitro GCs usingλ light chain-expressing B cells (λ B cells) from normal mice,carrier-primed T cells (CGG-T cells) from CGG immune mice, FDCs fromnormal mice, and ICs consisting of NP-CGG-anti-CGG. After overnightincubation, FDC-lymphocyte clusters were seen, resembling thosedescribed by Kosco et al (1992) (J. Immunol. 148, 2331-2339); theseclusters persisted through the 14 days of culture. It seems that naïve Bcells initially produced IgM; as indicated in FIG. 2A (4^(th) open bar),over 120 ng of IgM anti-NIP accumulated by day 7, using this combinationof immunogen and cells.

Anti-NIP is largely derived from λ-bearing B cells and the use ofpurified λ B cells was helpful as naïve B cells containing κ and λ Bcells (κ+λ B cells) in normal amounts (˜95% κ) did produce IgM anti-NIP(˜20 ng/mL), but not as well as the λ-bearing cells (FIG. 2, open bars 3vs. 4). Use of ICs that could be trapped and presented to B cells byFDCs was also important, as free antigen (NP-CGG) did not work as wellas ICs (FIG. 2, open bar 4 versus 5). If either immunogen (antigen orICs) or FDCs were missing or if OVA-primed T cells were substituted forCGG-primed T cells, the NIP-specific IgM response was typicallyundetectable. The low IgM response obtained with ICs in the absence ofFDCs at day 7 in this experiment was not a consistent observation. Theculture media were replaced on day 7 and some IgM accumulated in thesecond week (FIG. 2, solid bars), but the levels were low compared withanti-NIP IgM in the first week. The assay using NIP-5 to detect highaffinity antibody indicated very little IgM anti-NIP was produced, evenin the presence of FDCs (FIG. 2, panel B).

Example 13

Immunoglobulin class switching and NIP-specific IgG Responses in invitro GCs. The IgG anti-NIP-response was studied in the same culturesdescribed in FIG. 2 for the NIP-specific IgM. The need for λ B cells,CGG-primed T cells, FDCs, and NP-CGG-anti-CGG ICs was the same foroptimal IgG production and was apparent, as it was for IgM (FIG. 3A;3^(rd) open and 3^(rd) filled bars). The anti-NIP IgG that accumulatedin the first week in FIG. 3A was about half the level of anti-NIP IgM inFIG. 2A (˜60 ng/mL IgG versus ˜120 ng/mL IgM). However, theserelationships were reversed in the second week with over ˜140 ng/mL IgGversus only ˜20 ng/mL of IgM (filled bars in FIG. 2A versus 3A). Thus,the Ig isotype produced switched from predominantly IgM in the firstweek to predominantly IgG in the second week.

Example 14

In vitro affinity maturation detection and importance of FDC-ICs. Incontrast to IgM, large amounts of IgG were apparent when NIP-5 was usedto detect high affinity antibodies. Of interest, only about 30 to 50% ofthe IgG made in the first week was of high affinity (NIP-5 versusNIP-19). However, almost all of the IgG made in the second week was ofhigh affinity (FIG. 3B). This is consistent with selection of highaffinity B cells and selective stimulation of these cells to produce thehigh affinity IgG associated with affinity maturation. Furthermore,affinity maturation was only observed when antigen was in the form ofICs that would be trapped and presented to B cells by FDCs. Free antigen(NP-CGG) that should engage BCR efficiently did stimulate low affinityIgG (FIG. 3A, 1^(st) and 4^(th) filled bars) but did not stimulatedetectable levels of high affinity IgG (FIG. 3B). In the absence ofFDCs, ICs engage BCR and FcγRII leading to ITIM activation, SHIPphosphorylation, and a lack of responsiveness. Trapping the Ig-FC byhigh levels of FcgRII on FDCs minimizes engagement of FcgRII on the Bcell and facilitates a productive IgG response (Aydar et al. (2004) Eur.J. Immunol. 34, 98-107; Aydar et al. (2003) J. Immunol. 171, 5975-5987).Thus, it appears that the only ICs capable of stimulating B cells for aproductive IgG response are those trapped by the FDCs.

Example 15

Gauging the importance of FDC CD21-CD21 ligand interactions to IgMresponses and class switching. Interaction between FDC-CD21 ligand andCD21 in the B cell co-receptor complex (CD21/CD19/CD81) is important forFDC-associated antigen to stimulate optimal recall responses (Tew et al.(2001) Trends Immunol. 22, 361-36; Qin et al. (1998) J. Immunol 161,4549-4554). IgM responses in CD21/CD19 knockout mice are also depressed(Chen et al. (2000) Immunol. Rev. 176, 194-204). Thus, blocking signalsto B cells delivered via FDC-CD21 ligand-CD21 interactions may inhibitIgM production and class switching. Our results indicate that anti-CD21inhibited the IgM response and, consistent with a reduction in classswitching, the IgG response was dramatically reduced (>90%) at its peakin the second week. The diminished IgG response was not simplyattributable to a loss of B cells in the absence of CD21 ligand-CD21interactions because the number of B cells persisting in culturestreated with anti-CD21 was not significantly lower than the B cellnumber with the isotype control. We also considered the possibility thatthe Fc portion of the intact IgG binding B cell-CD21 could engage Bcell-FcγRII and lead to ITIM activation and thus explain the reducedantibody response with anti-CD21. However, if the anti-CD21 is simplyblocking the receptor then anti-CD21 F(ab′)₂ should work as well as theintact antibody and this proved to be the case.

Both FDCs and B cells express CD21 and CD23. CD23 is a ligand for CD21in the human system (Aubry et al. (1992) Nature 358:505), raising thepossibilities that anti-CD21 could influence FDC activity or thatFDC-CD23 could engage B cell CD21 and provide a signal to B cells.However, treating FDCs with anti-CD21 did not inhibit their activity andtreating B cells and FDCs with anti-CD23 did not have any detectableeffect.

We sought to determine whether increasing CD21 ligand levels on FDCswould increase class switching and production of high affinityNIP-specific IgG. Treating ICs with complement to enhance CD21 ligandlevels on the FDCs did not increase the anti-NIP response. This isconsistent with previous data where additional CD21 ligand did notincrease the murine anti-OVA response in normal mice (Aydar et al.(2002) Eur. J. Immunol. 32, 2817-2826). However, in aged mice the levelof CD21 ligand covalently bound to the FDCs appears to be low andaddition of rabbit complement to increase levels of FDC-CD21 ligand onaged FDCs improved accessory activity and the B cell responses (Aydar etal. (2002) Eur. J. Immunol. 32, 2817-2826).

Example 16

AID expression and the presence of FDCs. AID is important in classswitching and is expressed in GC-B cells and in B cells undergoing classswitch recombination in vitro (Muramatsu et al. (1999) J. Biol. Chem.274, 18470-18476; Muramatsu et al. (2000) Cell 102, 553-563; Faili etal. (2002) Nat. Immunol. 3, 815-821). FDCs may help regulate AIDexpression by GC-B cells; we sought to examine this. Expression of AIDmRNA can be detected in lymphocytes stimulated with LPS, or withIL-4+anti-CD40, where a large proportion of B cells are stimulated withthese polyclonal B cell activators. Costimulation of B cells with FDCsmight amplify AID mRNA. Quantitative RT-PCR was used to determine thelevels of AID mRNA. Suboptimal amounts of LPS (10 ng), IL-4 (10 ng), andanti-CD40 (100 ng) were used to stimulate low levels of AID in thelymphocytes. FDCs were added either at the beginning to providecostimulation or at the end of the culture so that mRNA coming from theFDCs would be constant in all cultures. AID mRNA level in normallymphocytes was defined as 1-fold to compare the effect of LPS, orIL-4+anti-CD40 treatment alone or in the presence of FDCs. Analysis withRT-PCR indicated that LPS increased AID in the lymphocyte populationabout 8-fold and FDCs about 2-fold. However, the combination of FDCswith LPS was synergistic and AID mRNA expression increased about130-fold. Results with IL-4+anti-CD40 were similar. The combination ofFDCs with IL-4+anti-CD40 resulted in AID mRNA levels up ˜180-fold versus˜3-fold with FDCs and about 18-fold with IL-4+anti-CD40.

No significant AID mRNA was found when FDCs alone were stimulated withLPS or anti-CD40+IL-4 suggesting that B cells were the source of themRNA when FDCs and B cells were cultured together. This was confirmed byisolating mRNA from B cells purified after the two-day culture periodusing B220 MicroBeads with the MACS system. Nearly all of the AID mRNAwas in the B cell fraction; while the flow-through fraction did containdetectable activity, it also contained some contaminating B cells,likely accounting for this AID mRNA. Furthermore, the increased AIDactivity in B cells did not appear to be simply attributable toincreased B cell survival or proliferation caused by FDCs, because thesame number of B cells (˜2×10⁶) was used to obtain the mRNA and the 18srRNA was used as an internal loading control. Thus, the level of AIDmRNA per B cell was elevated when B cells were cultured in the presenceof FDCs.

Example 17

CD21-CD21 ligand interactions involvement in AID expression and classswitching. The reduction in class switching observed when CD21ligand-CD21 interactions were blocked suggests that the interactionbetween FDC-CD21 ligand and B cell-CD21 might signal through theco-receptor complex and help regulate expression of AID. To examinethis, anti-CD21/35 was used to interrupt FDC-CD21 ligand-B cell-CD21interactions and the level of AID expression was reduced ˜90%,indicating that this interaction is playing a role.

Example 18

Importance of ICs and CD21 ligand for FDC-mediated enhancement of AIDresponses. We sought to determine whether ICs contribute to the abilityof FDCs to promote optimal high affinity antibody responses. Given theimportance of FDC-ICs in promoting class switching and affinitymaturation, we sought to determine whether ICs and CD21 ligand-CD21interactions were important in FDC-mediated enhancement of AIDexpression in the NP-CGG system. The small number of B cells respondingto NP-CGG makes the study of AID regulation more challenging. However,it was possible to detect FDC-mediated enhancement of AID mRNA when˜2×10⁶ purified B cells were used for RNA purification after 72 h ofculture. NP-CGG-anti-CGG ICs stimulated enhancement, while NP-CGG failedto stimulate detectable enhancement. Furthermore, anti-CD21/35 inhibitedthe antigen-stimulated response in the same fashion as was observed instudies of B cells stimulated with polyclonal activators.

Example 19

Somatic hypermutation in the in vitro GCs. To examine somatichypermutation in the in vitro GCs, we used PCR to amplify the VH186.2gene that is used in the mouse to make anti-NP. The PCR product was cutfrom an electrophoresis gel, extracted, and cloned; multiple clones werethen sequenced. 7 out of 20 readable sequences had homology to Vh186.2germline and were designated VH186 clones. The sequences have beenaligned against the VH186.2 germline-encoded gene. Mutations areindicated with the replaced nucleotide. As illustrated in FIG. 10,considerable mutation occurred in the variable gene, consistent withsomatic hypermutation. These mutations were more frequent in the CDRsequences (FIG. 11). Analysis of the mutations revealed:

-   -   an average of 41 mutations (range 32-45) were seen per VH186        gene (306 nucleotides) sequenced. This is high, consistent with        results obtained in studies of in vivo GCs.    -   most mutations were point mutations with one deletion. This is        also typical of in vivo germinal centers and somatic        hypermutation.    -   all mutations except one were replacement mutations in the CDRs        while the ratio of replacement to silent mutations in the        framework regions was almost 1:1, both indicating strong        selective pressure.    -   a predominance of transitions over transversions was observed.    -   we observed only one mutation in all the Cγ regions sequenced,        providing an internal control for the fidelity of PCR        amplification.

The mutational characteristics obtained in these in vitro GCs aresimilar to those observed in GCs in vivo for anti-NP responses. Thus,the in vitro GCs of the present invention appear to faithfully reflectimportant events that occur in GCs in vivo.

Example 20

CXCL 13, a chemokine secreted by FDCs has been shown to attract human Bcells and T cells into the follicular zones (Estes et al. (2004) J.Immunol. 173, 6169-6178). In other embodiments, blocking this chemokineor its receptor CXCR 5 may inhibit migration of B and T cells to theFDC-rich areas. Additionally, GC B cells are activated and express aunique phenotype, PNA⁺, GL-7⁺, CD95^(hi) and CD23^(lo) and segregateinto light zones where they are centrocytes and into dark zones wherethey are centroblasts. In other embodiments, these features can bepresent in the in vitro model of the lymph node follicle.

Example 21

In other embodiments, purified preparations of FDCs, B cells, or T cellscan be embedded into ETCs, including, e.g., cellulose-basedmicrocarriers, collagen cushions, and lymph node extracellularmaterials. This can be done by adding cells to the ETC suspensionsbefore they solidify or by directly injecting a suspension into theETCs. These cells can be allowed to equilibrate in the matrix and canthen be visualized and followed over a period of 2 weeks. Human B and Tcells can be isolated from peripheral blood of healthy donors bynegative selection using anti-14, -CD19, -CD3, and -CD56, to removeunwanted cells. Murine B and T cells can be obtained from lymph nodesand purified by positive selection, as previously described.

Example 22

As FDCs can co-stimulate B cells without MHC or species restriction, inembodiments of the present invention, FDCs can be isolated from eitherlymph nodes of naïve mice or human tonsils surgically removed from youngpatients, using the FDC-specific mAb HJ2, as previously described(Fakher et al. (2001) Eur. J. Immunol. 31, 176-185).

Example 23

In other embodiments, different procedures such as “in situjellification”, “injection”, “cushion-beads combinations”, pluscombinations with small cushions, a single cushion for all cell types,and perforations in the cushions, can be used for the general LTEarchitecture when incorporating FDCs. In other embodiments, the ETC caninclude, e.g., collagen cushions, cellulose based microcarriers,synthetic and other natural bio-materials, and/or lymph nodeextracellular materials.

Example 24

In further embodiments, FDCs, T cells, and B cells can be placed in thesame ETC but at different locations. The FDCs can be put in first andallowed to attach to ETC and then the T and B cells can be placed nearby. Lymphocytes will be attracted to the FDCs, where they can clusteraround the FDCs and form in vitro GCs. We have observed that theCD3-selected T cells and negative-selected B cells exhibit low cellmotilities in collagen cushions in the absence of chemokines. Thepresence of FDCs and associated chemokines may increase the naturalmotility of lymphocytes.

Example 25

In still other embodiments, FDCs, T cells, and B cells can be placed insingle ETCs to visualize clustering or in separate ETCs to simulate Tand B cell areas of a lymph node. In addition to regular microscopicanalysis, cells can be fluorescently labeled in the cushion andvisualized by confocal microscopy. Furthermore, in other embodiments, Band T lymphocytes isolated from tetanus toxoid (TT)-immunized personscan be co-cultured in these FDC-containing ETCs and further stimulatedwith TT-anti TT ICs, to serve as models for antigen-specific GCs.

Example 26

FDCs secrete the chemokine CXCL 13, which acts as a chemoattractant forboth B cells and follicular T helper cells, recruiting these cells intothe GC (54). In other embodiments, B and T cells can be added to theFDC-containing ETCs and stimulated using, e.g., LPS or Con A in thepresence or absence of neutralizing antibodies against CXCL13 or itsreceptor, CXCR5. In other embodiments, FDCs from CXCL13 knockout micecan be used. In other embodiments, anti-CD21, anti-ICAM-1, anti-VCAM,and anti-BAFF antibodies can be separately added to these cultures toexamine the importance of these surface molecules in the formation ofFDC-B cell-T cell clusters. Previous studies have indicated a role forthese molecules in the clustering of B cells around FDCs in culturewells.

Example 27

Characteristics of GCs and inclusive B cells when FDCs are loaded withantigen. GCs are formed about 6 to 8 days after primary antigenchallenge and are detected by the presence of FDCs decorated with ICsand complement fragments in the light zones present next to dark zonesconsisting of rapidly dividing B cells expressing the unique GC B cellphenotype (PNA⁺, GL-7⁺, CD95^(hi), CD23^(lo)). These GC B cells are Bcells responding to the specific antigen and undergo class switching andsomatic hypermutation, generating IgG antibodies of higher affinity.

Example 28

In other embodiments, B and T cells isolated from TT-immunizedindividuals can be cultured in collagen cushions containing FDCs andstimulated with TT-anti TT ICs. After about 10 days in culture, B cellscan be harvested and labeled and analyzed for surface expression of PNA,GL-7, CD95 and CD23 by flow cytometry. Detailed analysis of themorphology of these follicles can also be made using confocalmicroscopy.

Example 29

In still other embodiments, B and T cells isolated fromantigen-immunized individuals can be cultured in collagen cushionscontaining FDCs and stimulated with antigen-anti antigen ICs. Afterabout 10 days in culture, B cells can be harvested and labeled andanalyzed for surface expression of PNA, GL-7, CD95 and CD23 by flowcytometry. Detailed analysis of the morphology of these follicles canalso be made using confocal microscopy.

Example 30

In the 2D clusters we have studied in vitro, we have not seen B cellsforming a mantle area around the GC or T cells, collecting together toform a separate unit. However, the 2D arrangement has no reticularfibers or other structures to help arrange the cells. In otherembodiments of the present invention, ETCs can be used to provideconditions such that T cells and B cells segregate around the GC andform distinct areas. The cells surrounding the GCs can be examined usingT cell- and B cell-specific antibodies. Labeling with anti-IgD may alsobe informative because GC B cells do not express IgD, whereas mantlezone B cells do.

When secondary lymphoid tissues are challenged with appropriate antigensthey expand dramatically, as a consequence of large numbers of newfollicles developing with active GCs. These follicles are organized withdistinct T cell zones and B cell zones and an active GC consists ofrapidly proliferating B cells, helper T cells, FDCs, and macrophagescleaning up apoptotic B cells that have not received survival signalsfrom FDCs. Each active GC is further divided into a light zone whereFDCs, T cells, and B cells are interacting and a dark zone where the Bcells are rapidly proliferating.

Follicles are apparent not only in lymph nodes but in spleens, lymphoidnodules (e.g., Peyer's patches) and are conserved in all species withlymph nodes. It does not appear to matter whether the antigen is broughtinto the follicle from the afferent lymph, as it is in a lymph node, orfrom the blood via the marginal sinus of the spleen or by M cells (as isthe case in Peyer's patches).

Once antigen is in the follicle, the agonist necessary for developmentof a secondary follicle with an active GC and development of humoralimmunity is present. The follicular structure, fully developed in 3D, islikely important to the production of adequate amounts of high affinityantibody.

Example 31

When lymphoid tissues are digested and the cells are placed intoconventional tissue culture they lose the follicular organization andremain disorganized in 2D. In embodiments of the present invention, FDCscan be added to such cultures to form FDC-B cell-T cell clusters. Wehave demonstrated immunoglobulin class switching, somatic hypermutation,and affinity maturation in such in vitro GCs.

Example 32

In embodiments of the present invention, follicular leukocytes can beplaced in an ETC matrix, natural or synthetic, where FDCs can be fixedand T and B cells can arrange themselves around the FDCs to recreateaspects of the in vivo environment of the follicle. Suitable materialsfor the ETC include collagen, gelatin, hyaluronic acid, extracellularmatrix (ECM), small intestine submucosa, urinary bladder mucosa, PLGA,hydrogels, inverted colloid crystal matrices, microcarriers, and platescoated with collagen. In other embodiments of the present invention,there is no ETC matrix; the T cells, B cells, and FDCs are simplycultured in standard, 2D wells.

Example 33

In other embodiments of the present invention, a functional LTEcontaining FDCs and established or non-established T and B cell zonescan be used to assess vaccines. The FDCs can be used to assist inestablishing the T and B cell zones; they do not need to be pre-formed.Likewise, in another embodiment, the T and B cell zones are notrequired. In primary immune responses, monocyte-derived dendritic cellsprimed from the integrated in vitro vaccination site can be used, Modelantigens, such as tetanus for a recall response and influenza tovalidate the model in a primary response, can be used, as can otherantigens, immunogens, and/or allergens.

Example 34

In studies using murine cells, we present evidence for a functional invitro GC in 2D culture. Specifically, murine GCs were set up in vitro byco-culturing naïve λ-positive B cells, FDCs, NP-CGG (chickengammaglobulin)+anti-CGG ICs, and CGG-primed T cells. This resulted inFDC-lymphocyte clusters and production of anti-NIP IgM and IgG.

Class switching was indicated by a shift from IgM in the first week toIgG in the second week and affinity maturation was indicated by a changefrom mostly low affinity IgM and IgG in the first week to virtually allhigh affinity IgG anti-NIP in the second week. Class switching andaffinity maturation were easily detectable in the presence of FDCsbearing appropriate immune complexes (ICs) but not in the absence ofFDCs or FDCs with irrelevant antigens in ICs.

Free antigen plus FDCs resulted in low affinity IgG, but affinitymaturation was only apparent when FDCs bore ICs. Class switching isactivation-induced cytidine deaminase (AID)-dependent and blockingFDC-CD21 ligand-B cell CD21 interactions inhibited FDC-IC-mediatedenhancement of AID production and the IgG response. FDCs promoted theproduction of both AID and error-prone polymerases; these enzymes areneeded for somatic hypermutations. Sequencing of the variable regiongenes indicated large number of mutations consistent with the productionof high affinity antibodies.

Example 35

In an embodiment of the present invention, a human cell system in a 3Dengineered tissue construct is constructed that can be integrated in anAIS. The FDCs can attach to collagen fibers, which provide a scaffold inwhich GCs can develop in 3D, as they would in vivo. NIP-specific IgM andIgG production, class switching, somatic hypermutation and affinitymaturation established in the murine system can be compared with theresults from previous studies using the 2D in vitro GCs.

Example 36

In an embodiment of the present invention, human GCs can be establishedusing tetanus toxoid (TT) as a carrier, because TT-specific memory Tcells are abundant in most people. In other embodiments, NP-tetanustoxoid can be used to set up the GCs. Anti-NP production can then beexamined as this allows simple determination of the production of bothhigh and low affinity antibodies and allows examination of affinitymaturation. In other embodiments naïve human T cells can be primed invitro and then used to provide T cell help for the in vitro GCs. Naïvehuman T cells can be primed with CGG, using monocyte-derived DCs fromeither culture or from in vitro vaccination site (VS) cells pulsed withCGG. Such primed T cells can then be used in the same way as used forthe murine system with CGG-NP.

Example 37

FDCs are attached to reticular fibers in vivo and are in immobilenetworks in the follicles. The lymphocytes recirculate but the FDCs arestationary. However, the FDCs in this condition in vivo have matured.Collagen cushions made from rat-tail collagen were established. FDCsattached and set up clusters on the collagen.

Example 38

The inclusion of immune complexes in the in vitro LTE is important forthe generation of fully differentiated memory B cells. In an embodimentof the present invention, a two-stage LTE is used. In the first step,naïve antigen-specific B cells are stimulated to produce antibody in a Tcell-dependent manner. Immune complexes and memory T cells elicited fromthis construct, in concert with FDC, provide the signals to trigger afresh batch of naïve B cells to fully differentiate in the second LTEconstruct.

In another approach, ICs can by generated by artificially couplingantibody to antigen in a non-specific manner. For example, a hapten isconjugated to the antigen of interest, which is then be bound by aspecific antibody. Fluorescein isothiocyanate (FITC) can be linked toprimary amino groups of target protein using literature procedures, withspecial attention taken to retain the antigenicity of the protein afterconjugate formation. In this regard, Fluorescein-EX or other derivativesbearing elongated linkers may be advantageous over tight linker-antigenconjugates formed by FITC and other haptens. Commercially availablehigh-affinity anti-FITC antibodies can then be used to bind theantigen-hapten conjugate, forming a complete IC. Tetanus toxoid can beused as a model antigen because most adults have immunized been with itand humoral and cell-mediated immune responses generated against thisantigen are well characterized. Other linkers and antigens, such asdigoxin and NP, respectively, can also be used. In another embodiment,the antibody can be chemically coupled directly to the antigen using theamine-thiol cross-linking method. Using these non-specific chemistriesdoes not require an agglutination step, making them useful forpolyclonal antibodies. Additionally, the stoichiometry of the IC can bemanipulated without affecting the size or density of this complex.

Example 39

In this example, an experiment was conducted, adding LPS to B cells toprovide a signal and then adding FDCs to provide a costimulatory signal.Antibody production was then examined. We followed the cells for 2weeks. Culture media were collected at the end of the first week and thetiters represent antibody synthesis from days 1 to 7. The media werereplaced and the supernatant fluids were collected at day 14; thosetiters represent antibody synthesis from days 7 to 14. FDCs had potentcostimulatory activity, as expected but at 7 days there was nodifference in antibody production between FDCs attached to collagen andFDCs floating on the plastic plate. However, at day 14 the FDCs attachedto the collagen were about three times as active in promoting antibodyproduction when compared with those floating on the plastic plates. TheIgG response in the second week was lower than the first week but thisis typical for LPS-stimulated cells, which respond rapidly and taper offrapidly. In contrast, antigen-stimulated cells typically reach a peak inIgG production in the second week. These results demonstrate thatputting FDCs on collagen enhances biological activity.

Example 40

FIG. 12 illustrates freshly isolated FDCs. A few FDCs can be found withtypical processes before positive selection using the monoclonalantibody, FDC-M1. However, after positive selection, few processespersist (FIGS. 13, 14, 15).

Example 41

We sought to induce primary human IgG responses in vitro and to generatehigh quality antibodies with an affinity that will enable them tofunction at low concentrations.

Ovalbumin (OVA) was used as an example antigen; the block donor used wasOVA-seronegative. T cells were primed with monocyte derived DCs.Monocytes (˜1×10⁷) were cultured with IL-4 (˜1000 U/mL) and GM-CSF (˜800U/mL) to generate immature DCs. After 5 d, OVA (1 μg/mL) was added toprovide antigen for processing and LPS (1 μg/mL) was added to stimulateDC maturation. After 8 h, ˜20×10⁶ CD4⁺ T cells were added for OVApriming. The priming and maturation for helper T cells was allowed tocontinue for 5 d in the mouse experiment (experiment 1) and 10 d(experiment 2). After this priming period, the T cells and DCs weremixed with naïve B cells (˜15×10⁶) in experiment 1 and 10×10⁶ inexperiment #2. OVA (˜5 μg)+murine anti-OVA (˜30 μg) were complexed togenerate OVA ICs and the cells and ICs were injected behind the neck ofirradiated mice for experiment #1 and the ICs were placed in vitro with˜3×10⁶ freshly isolated FDCs for experiment #2. Thus, in experiment 1 weobtained ˜30 ng/mL of anti-OVA at day 14.

In experiment 2, at day 5, we measured specific anti-OVA at ˜12 ng/mL.At day 10 the anti-OVA levels were at ˜20 ng/mL. (these levels werereadily assessed by ELISA). Thus, in 50 mL of media, this corresponds to˜600 ng of anti-OVA at day 5 and ˜1000 ng of total anti-OVA for day 10.We next tested for affinity maturation. The test was to let the anti-OVAbind to the ELISA plate and then to add a high salt concentration andquantitate how much of the bound antibody dissociates over 2 h on ashaker. The plates were then washed and the ELISA was conducted asbefore. Low affinity antibody will dissociate and be washed away andhigh affinity antibody will remain bound and detected in the ELISA. Mostof the day 5 antibody dissociated with 1 M NaCl and most of the day 10antibody remained bound, implying that over that time period there was achange from low to high affinity antibodies.

Example 42

Total mouse IgG produced by fresh murine FDCs on collagen type I beadsafter incubation with murine lymphocytes and LPS and with or without ICand complement. In this example, we sought to activate the FDCs byadding fresh immune complexes and complement after they had set upnetworks on collagen. We measured ˜8-9000 ng of antibody with freshFDCs, After 7 days, FDCs with ICs, the antibody concentration was still˜8-9,000 ng/mL. After 14 d, the antibody concentration was 4-5000 ng/mLwith ICs and FDCs maintained on the beads being better than thosemaintained on plastic. Thus, the FDCs may be maintained with goodactivity on beads for at least about 2 wk. Human FDCs benefited from ICsand complement.

Example 43

Direct deposition of collagen without chemical crosslinking. In thisexample, PuraCol (ultra-pure bovine Type I collagen in 1 mM nitric acid,Inamed, Calif.) solution (˜50 μL) was placed in a 250 μL plastic pipettetip and used as a ‘pen’ for manual patterning, without a pipette. Thismethod allowed the printing of oval and circular spots (˜800-1000 μm)that were able to endure washing and incubation with a cell culture. Thetissue culture plates or Petri dishes thus spotted with collagen wereplaced in a biohazard hood and dried for ˜1 h under the UV light of thehood. The tissue culture (multi-well) plates or Petri dishes patternedwith collagen as described were filled with PBS and incubated at roomtemperature for ˜10 min. The dishes were then emptied, filled withdistilled water and incubated for ˜5 min. This distilled water wash wasrepeated three times, after which the dishes were dried in the biohazardhood for 3 h, including ˜30 min further exposure to the UV light (FIG.16).

Example 44

Deposition of collagen with chemical crosslinking. In anotherembodiment, to increase hardiness of the collagen spots, methods ofchemical crosslinking can be used. As an example, glutaraldehyde can beadded to the washing/neutralizing solution to initiate crosslinking ofthe collagen. Unreacted glutaraldehyde can be neutralized by washingwith solution of trimethylamine, and removed via multiple washes withdistilled water.

Example 45

Patterning by laser micromachining. In another embodiment, continuouscoating of the dishes with collagen and subsequent patterning usinglaser beam is a method that can be used to create regular patterns.Laser micromachining can also be useful in the chemical modification andactivation of the plastic surfaces that would improve attachment andstability of the collagen patterns.

Example 46

Memory B cells are formed in large numbers in germinal centers in vivo.They are also found in the in vitro germinal centers of the presentinvention. To assess this, we took human lymphocytes 12 d after thestart of the in vitro primary and added fresh FDCs and immune complexesto provoke a secondary response. Supernatant fluids from these cultureswere collected and contained measurable levels of high affinity specificIgG antibody.

Example 47

In an embodiment of the present invention, one can induce primaryresponses against dangerous immunogens in vitro and expand the specificmemory cells in an in vitro GC and then use these memory cells with moreFDCs and immunogen to further expand the cultures. This process can berepeated one or more times. The final product is large amounts ofhigh-affinity, specific human IgG antibodies without exposing a human tothe dangerous immunogen.

Example 48

FIG. 16 illustrates collagen dots prepared according to Example 43. Theresults in FIG. 17 are with collagen-coated Cytodex beads and the ˜7,000ng of IgG/mL with FDCs on Cytodex is a typical result. The results inFIG. 18 are with the collagen dot pattern. There, we measured ˜47,000 ngof IgG.

An embodiment of the present invention comprises having FDCs adhered tothe collagen dots, where they attract lymphocytes. The collagen dots canbe prepared with, for example, bovine collagen or rat tail type 1collagen (FIG. 16). The collagen can also be used to cover the base oftissue culture plate wells, for example. A high level of antibodyresulted (˜29,500 ng/mL).

Without wishing to be bound by any mechanism, it seems that some FDCsstick to the top of the collagen dots, while others form a ring aroundthe bottom of the dots. Those sticking to the top form irregular shapedclusters and appear to attract lymphocytes; the lymphocytes become moredispersed further away from the FDC network.

While the foregoing specification teaches the principles of the presentinvention, with examples provided for the purpose of illustration, itwill be appreciated by one skilled in the art from reading thisdisclosure that various changes in form and detail can be made withoutdeparting from the true scope of the invention.

1. A method of evaluating an immune response to an antigen, comprising:a) administering a selected antigen to an in vitro cellular system,wherein the in vitro cellular system comprises a culture comprising anengineered tissue construct, and at least one three-dimensionalartificial germinal center embedded in or fixed on the engineered tissueconstruct, wherein the artificial germinal center comprises a pluralityof follicular dendritic cells, a plurality of B cells, and a pluralityof T cells; and b) evaluating B cell, T cell, or both B cell and T cellresponses to said antigen.
 2. The method of claim 1, wherein saidantigen is selected from the group consisting of a vaccine, an adjuvant,an immunomodulator, an immunotherapy candidate, a cosmetic, a drug, abiologic, and a chemical compound.
 3. The method of claim 1, whereinsaid antigen is coupled with an antibody specific for said antigen. 4.The method of claim 1, wherein said engineered tissue construct isselected from the group consisting of a collagen cushion, gelatin,hyaluronic acid, small intestine submucosa, urinary bladder mucosa,PLGA, a hydrogel, and a tissue culture plate coated with collagen,microcarriers, inverted colloid crystal matrices, syntheticextracellular matrix materials, or natural extracellular matrixmaterials.
 5. The method of claim 1, wherein said B cells and T cellsare isolated from an individual immunized with an antigen selected fromthe group consisting of a vaccine, an adjuvant, an immunomodulator, animmunotherapy candidate, a cosmetic, a drug, a biologic, and a chemicalcompound.
 6. The method of claim 1, wherein said culture furthercomprises follicular leukocytes.
 7. The method of claim 1, wherein saidengineered tissue construct comprises a tissue culture plate coated withcollagen spots.
 8. The method of claim 7, wherein said collagen spotsare crosslinked.
 9. The method of claim 7, wherein said folliculardendritic cells are adhered to the collagen spots.
 10. The method ofclaim 1, wherein said in vitro cellular system further comprises stromalcells distributed in the engineered tissue construct.
 11. The method ofclaim 1, wherein said culture further comprises soluble factors selectedfrom the group consisting of IL-4, CD40L, and anti-CD40 antibodies. 12.The method of claim 1, wherein a T cell response to the antigen isevaluated.
 13. The method of claim 1, wherein a B cell response to theantigen is evaluated.